VSX2 and ASCL1 Are Indicators of Neurogenic Competence in Human Retinal Progenitor Cultures

<div><p>Three dimensional (3D) culture techniques are frequently used for CNS tissue modeling and organoid production, including generation of retina-like tissues. A proposed advantage of these 3D systems is their potential to more closely approximate <i>in vivo</i> cellular microenvironments, which could translate into improved manufacture and/or maintenance of neuronal populations. Visual System Homeobox 2 (VSX2) labels all multipotent retinal progenitor cells (RPCs) and is known to play important roles in retinal development. In contrast, the proneural transcription factor Acheate scute-like 1 (ASCL1) is expressed transiently in a subset of RPCs, but is required for the production of most retinal neurons. Therefore, we asked whether the presence of VSX2 and ASCL1 could gauge neurogenic potential in 3D retinal cultures derived from human prenatal tissue or ES cells (hESCs). Short term prenatal 3D retinal cultures displayed multiple characteristics of human RPCs (hRPCs) found <i>in situ</i>, including robust expression of VSX2. Upon initiation of hRPC differentiation, there was a small increase in co-labeling of VSX2+ cells with ASCL1, along with a modest increase in the number of PKCα+ neurons. However, 3D prenatal retinal cultures lost expression of VSX2 and ASCL1 over time while concurrently becoming refractory to neuronal differentiation. Conversely, 3D optic vesicles derived from hESCs (hESC-OVs) maintained a robust VSX2+ hRPC population that could spontaneously co-express ASCL1 and generate photoreceptors and other retinal neurons for an extended period of time. These results show that VSX2 and ASCL1 can serve as markers for neurogenic potential in cultured hRPCs. Furthermore, unlike hESC-OVs, maintenance of 3D structure does not independently convey an advantage in the culture of prenatal hRPCs, further illustrating differences in the survival and differentiation requirements of hRPCs extracted from native tissue vs. those generated entirely <i>in vitro</i>.</p></div

VSX2+ hRPCs are abundant within short and long term cultures of 3D optic vesicles derived from hESCs.

<p>20 days after initiation of retinal differentiation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135830#pone.0135830.ref020" target="_blank">20</a>], WA09 hESCs formed optic vesicle structures (OVs) comprised of VSX2+ hRPCs co-expressing (<b>A</b>) KI67, (<b>B</b>) NESTIN, and (<b>C</b>) SOX2. (<b>D-F</b>) At day 50, the majority of VSX2+ hRPCs remained KI67+. <b>(G)</b> VSX2+/KI67+ progenitors were also present at day 50 in hESC-OVs derived from the WA01 line. 50 day VSX2+ hRPCs continued to express (<b>H</b>) NESTIN and (<b>I</b>) SOX2. (<b>J</b>) At 20, 50, and 90 days of differentiation, the percentages of VSX2+ or KI67+ cells and (<b>K</b>) VSX2+ cells co-labeled with other progenitor markers were quantified. Nuclei were visualized with DAPI and cell count data is expressed as % immunopositive cells. Scale bar: 50 μm (panels A-D,H,I); 20 μm (panels E,F,G).</p

Neurogenesis decreases over time in human prenatal retinal neurosphere cultures.

<p>VSX2 immunoreactivity is not associated with (<b>A</b>) RECOVERIN+ cells in prenatal retinal tissue but does co-label a small subset of (<b>B</b>) βIII TUBULIN+ neurons. A similar pattern of VSX2 co-expression with (<b>C</b>) RECOVERIN and (<b>D</b>) βIII TUBULIN is observed in short term prenatal retinal neurosphere cultures; <i>arrow</i> in panels B and D indicate VSX+/βIII TUBULIN+ cells. (<b>E</b>) A subpopulation of PKCα+ cells also co-labels with VXS2 (<i>arrow</i> and <i>arrowhead</i> in panel E indicate VSX+/PKCα+ and VSX2-/PKCα+ cells, respectively). (<b>F</b>) The percentage of cells expressing VSX2 and selected neuronal markers were quantified in short term cultures. (<b>G</b>) Human prenatal retinal neurosphere cultures (n = 5) from day 79–108 gestation tissue were sampled at 1 week, 1 month, and 2 months, and the number of cells immunolabeled with selected neuronal markers was quantified. Nuclei were visualized with DAPI and cell count data is expressed as % immunopositive cells. ND: nondetectable. Scale bars: 50 μm (panels A,B); 20 μm (panels C-E).</p

NOTCH inhibition augments production of PKCα+ neurons in short term prenatal human retinal neurospheres.

<p>The percentages of cells immunopositive for (<b>A</b>) ASCL1 and (<b>B</b>) other selected hRPC and neuronal markers following DAPT treatment was determined by immunocytochemistry. (<b>C</b>) RT-PCR analysis was used to evaluate expression of VSX2 and ASCL1 in prenatal human retinal tissue and short (1 week) and long (2 month) term prenatal retinal neurosphere cultures. (<b>D</b>) Phase photomicrographs of dissociated prenatal retinal neurospheres reveal profound cell morphology differences between short term and long term cultures. Cell counts are expressed as % immunopositive cells. *p<0.05; **p<0.01. Scale bars in panel E: 50 μm.</p

Short term cultures of human retinal neurospheres retain a robust population of VSX2+ proliferating progenitor cells from source prenatal retinal tissue.

<p>VSX2+/KI67+ proliferating hRPCs were observed in the outer neuroblastic layer of the developing retina at (<b>A</b>) 59 days, (<b>B</b>) 73 days, (<b>C</b>) 85 days, (<b>D</b>) 93 days, and (<b>E</b>) 108 days of gestation. (<b>F-I</b>) VSX2+/KI67+ co-labeled cells were also present in dissociated cells from short term prenatal retinal neurospheres established from retinal tissue of similar gestational ages. (<b>J</b>) Short term prenatal retinal neurospheres were dissociated and immunostained to determine the percentage of cells expressing VSX2 and/or KI67. Nuclei were visualized with DAPI. The insert is a 4X magnification of the indicated area in panel A. Scale bars: 100 μm (panel A); 50 μm (panels B-E); 20 μm (panels F-I).</p

Prenatal retinal neurospheres lose VSX2 expression over time in culture.

<p>KI67+ hRPCs in the outer neuroblastic layer of 96 day human prenatal retina co-express the neural stem cell markers (<b>A</b>) NESTIN and (<b>B</b>) SOX2. Short term prenatal retinal neurosphere cultures also contain abundant (<b>C</b>) KI67+/NESTIN+ and (<b>D</b>) KI67+/SOX2+ hRPCs. Nearly all VSX2+ hRPCs in short term prenatal retinal neurosphere cultures co-label with (<b>E</b>) NESTIN and (<b>F</b>) SOX2. Prenatal retinal neurosphere cultures (n = 5) from 79–108 day gestation tissue were sampled at 1 week, 1 month, and 2 months. After 2 months, very little VSX2 immunostaining is detected, although (<b>G</b>) NESTIN and (<b>H</b>) SOX2 remain highly expressed. The percentage of VSX2, KI67, NESTIN, and SOX2 immunopositive cells were quantified (<b>I</b>) in short term cultures and (<b>J</b>) over a 2 month period. Nuclei were visualized with DAPI and cell count data is expressed as % immunopositive cells. Scale bars: 50 μm (panels A,B); 20 μm (panels C-H).</p

VSX2+ hRPCs from 3D hESC-OVs maintain neurogenic competence and continue along a normal developmental trajectory in long term cultures.

<p>VSX2+/ ASCL1+ hRPCs were detected in 3D hESC-OVs at (<b>A</b>) 30 days, (<b>B</b>) 50 days, and (<b>C</b>) 90 days of differentiation. (<b>D</b>) The percentages of ASCL1+ cells that co-labeled with VSX2 were quantified in 30, 50, and 90 day hESC-OVs. (<b>E</b>) By 50 days, many photoreceptor precursor cells identified by RECOVERIN immunoreactivity were present in hESC-OVs, and by 90 days (<b>F,G</b>), RECOVERIN+ cells increased in abundance. (<b>H</b>) Quantification of cells expressing ASCL1 or RECOVERIN was performed at 20, 30, 50, and 90 days of differentiation. Nuclei were visualized with DAPI and cell count data is expressed as % immunopositive cells. ND: nondetectable. <i>Arrows</i> in panels A-C demarcate ASCL1+/VSX2+ cells. Scale bars: 50 μm (panels E,F); 20 μm (panels A-C,); 10 μm (panel G).</p